The Heat shock protein 70 (Hsp70) family members have broad chaperone functions in cells that include folding of nascent proteins, refolding of misfolded proteins, removal of protein complexes, and control of regulatory proteins [Daugaard, M., Rohde, M., and Jaattela, M. (2007). The heat shock protein 70 family: Highly homologous proteins with overlapping and distinct functions. FEBS letters 581, 3702-3710; Evans et al. (2010). Heat shock protein 70 (hsp70) as an emerging drug target. Journal of medicinal chemistry 53, 4585-4602]. These functions are driven by ATP hydrolysis in the N-terminal nucleotide-binding domain (NBD) of the Hsp70s. The Hsp70s are evolutionary conserved across species and there are 8 mammalian family members [Hunt, C., and Morimoto, R. I. (1985). Conserved features of eukaryotic hsp70 genes revealed by comparison with the nucleotide sequence of human hsp70. Proceedings of the National Academy of Sciences of the United States of America 82, 6455-6459]. The inducible form of Hsp70 (Hsp70i, also called Hsp72, Hsp70-1, HspA1A/HspA1B) is present in low or undetectable levels in unstressed normal cells, however, expression levels rapidly increase in response to cellular stresses such as heat shock, viral infection or transformation. Deletion of its immediate paralog, the constitutive heat shock protein cognate 70 (Hsc70) is developmentally lethal, whereas deletion of Hsp70i results in sterility of male mice, but no other overt phenotype [Dix et al. (1996). Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility. Proceedings of the National Academy of Sciences of the United States of America 93, 3264-3268; Wacker et al. (2009). Loss of Hsp70 exacerbates pathogenesis but not levels of fibrillar aggregates in a mouse model of Huntington's disease. The Journal of neuroscience: the official journal of the Society for Neuroscience 29, 9104-9114]. Hsp70i and Hsc70 are highly related, sharing 90% sequence identity, with most of the amino acid variability confined to the NBD. There is greater sequence divergence with respect to other Hsp70 family members (50-80% identity), especially within the NBD [Daugaard et al., 2007]. The close sequence similarity between Hsp70i and Hsc70 has contributed to past difficulties in separating the two proteins' functions, both pharmacologically and with RNA interference approaches.
Overexpression of Hsp70i has been observed in several cancers, including breast, prostate, and colon, and this is thought to aid in resistance to apoptosis as well as to anti-cancer treatments [Goloudina et al. (2012). Inhibition of HSP70: a challenging anti-cancer strategy. Cancer letters 325, 117-124; Shu et al. (2008). HSP70s: From Tumor Transformation to Cancer Therapy. Clinical medicine Oncology 2, 335-345]. Hsp70i inhibits both intrinsic and extrinsic apoptosis pathways. This occurs by preventing TNF-related apoptosis-inducing ligand formation of the death-induced signaling complex through inhibition of death receptors 4 and 5, as well as by inhibiting events in mitochondrial-mediated apoptosis [Goloudina et al., 2012; Guo et al. (2005b). Mechanistic role of heat shock protein 70 in Bcr-Abl-mediated resistance to apoptosis in human acute leukemia cells. Blood 105, 1246-1255]. In the latter case, Hsp70i prevents Bax translocation to the mitochondria, preventing release of cytochrome c, an apoptosis inducing factor [Yang et al. (2012). Hsp70 promotes chemoresistance by blocking Bax mitochondrial translocation in ovarian cancer cells. Cancer letters 321, 137-143]. Additionally, Hsp70i mediates both caspase dependent and independent apoptotic pathways by binding Apaf-1, blocking recruitment of procaspase-9 to the apoptosome, and by inhibition of JNK, respectively [Beere et al. (2000). Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome. Nature cell biology 2, 469-475; Park et al. (2001). Hsp72 functions as a natural inhibitory protein of c-Jun N-terminal kinase. The EMBO journal 20, 446-456]. Hsp70i also protects cancer cells from oncogenic stress induced by up-regulation of specific oncogenes such as HER2 [Afanasyeva et al. (2007). Drug-induced Myc-mediated apoptosis of cancer cells is inhibited by stress protein Hsp70. International journal of cancer Journal international du cancer 121, 2615-2621]. Increased expression of Hsp70i correlates with resistance to chemotherapy and radiation and therefore poor clinical outcomes by providing cancer cells a route to survive and proliferate in the presence of noxious stimuli such as hypoxia or denatured protein aggregates [Jego et al. (2013). Targeting heat shock proteins in cancer. Cancer letters 332, 275-285]. These data have led to the proposal that cancer cells are dependent on Hsp70i for survival [Goloudina et al., 2012]. This hypothesis is supported by Hsp70i depletion studies in which tumor cell death and sensitivity to chemotherapeutic drugs were evident, while non-tumorigenic cell lines were unaffected by Hsp70i depletion [Nylandsted et al. (2002). Eradication of glioblastoma, and breast and colon carcinoma xenografts by Hsp70 depletion. Cancer research 62, 7139-7142].
From a drug discovery perspective, Hsp70i presents a number of challenges, not least of which being its close sequence identity with Hsc70. Specific, physiological substrates of Hsp70i are poorly defined and high throughput assays based on chaperone or trafficking activities are limited [Kang et al. (2008). Design of a fluorescence polarization assay platform for the study of human Hsp70. Bioorganic & medicinal chemistry letters 18, 3749-3751]. The crystal structure of Hsp70i shows the protein in either a closed nucleotide bound state or open unbound state [Qi et al. (2013). Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP. Nature structural & molecular biology 20, 900-907]. In the closed conformation, the bound nucleotide shows little solvent accessibility to the surface, limiting access to diffusible small molecule inhibitors. In cells, Hsp70s may be reminiscent of small G proteins in which the nucleotide-binding pocket is always occupied, undergoing GTP/GDP exchange upon activation, again limiting small molecule accessibility. In the case of Hsp70i, the protein has high affinity for ADP, which is likely exchanged with ATP through allosteric regulation [Powers et al. (2010). Targeting HSP70: the second potentially druggable heat shock protein and molecular chaperone? Cell Cycle 9, 1542-1550; Swain et al. (2007). Hsp70 chaperone ligands control domain association via an allosteric mechanism mediated by the interdomain linker. Molecular cell 26, 27-39]. The chaperone activities of Hsp70i are also regulated by the C-terminus in cooperation with co-chaperones, such as Hsp40, Hip, Hop, CHIP and Bag1 [Tavaria et al. (1996). A hitchhiker's guide to the human Hsp70 family. Cell stress & chaperones 1, 23-28]. Crystallographic and NMR studies have shown that these co-chaperones induce altered conformational states [Evans et al., 2010; Mayer et al. (2005). Hsp70 chaperones: cellular functions and molecular mechanism. Cellular and molecular life sciences: CMLS 62, 670-684]. Because of these many complications, most Hsp70 inhibitors have either failed to discriminate between various Hsp70 family members or perform poorly in vivo [Massey, A. J. (2010). ATPases as drug targets: insights from heat shock proteins 70 and 90. Journal of medicinal chemistry 53, 7280-7286].
Prior inhibitors identified to target Hsp70s include NSC 630668-R/1, VER, MAL3-101, MKT-077, PES, Apoptozole, and YK5 [Powers et al., 2010; Rodina et al. (2013). Identification of an allosteric pocket on human hsp70 reveals a mode of inhibition of this therapeutically important protein. Chemistry & biology 20, 1469-1480]. There is considerable structural diversity amongst these inhibitors and generally the NBD domain has been favored for inhibitor development [Powers et al., 2010]. However, the polar interactions present in the nucleotide binding pocket and its affinity for ATP have contributed to difficulties in selective inhibitor discovery [Massey, 2010]. The full-length crystal structure of the nucleotide bound form shows that the nucleotide is completely enclosed, making the accessibility of small inhibitors difficult (FIG. 1B). Approaches adopted thus far have not been able to target specific Hsp70 family members, especially Hsp70i from Hsc70. NSC 630668-R/1, inhibits ATPase activity but does not discriminate Hsp70i from Hsc70 [Fewell et al. (2001). Identification of an inhibitor of hsc70-mediated protein translocation and ATP hydrolysis. The Journal of biological chemistry 276, 910-914]. VER shows broad specificity with other Heat shock protein family members, largely because it is a nucleotide derivative. It also contains two potentially labile, perhaps by design, benzyl groups [Massey, 2010]. MAL3-101 has been shown to compromise co-chaperone-stimulated Hsp70 ATPase activity, suggesting it is an allosteric regulator, although the exact binding site of this molecule remains unknown [Braunstein et al. (2011). Antimyeloma Effects of the Heat Shock Protein 70 Molecular Chaperone Inhibitor MAL3-101. Journal of oncology 2011, 232037]. Like NSC 630668-R/1, MAL3-101 is quite large and has a number of labile ester groups. MKT-077 targets the NBD and inhibits proliferation in tumor cell lines, however, severe renal dysfunction in patients was observed in phase I clinical trials [Britten et al. (2000). A phase I and pharmacokinetic study of the mitochondrial-specific rhodacyanine dye analog MKT 077. Clinical cancer research: an official journal of the American Association for Cancer Research 6, 42-49]. PES has been shown to interact with the SBD of both Hsc70 and Hsp70i and disrupt client protein interaction in vitro [Leu et al. (2009). A small molecule inhibitor of inducible heat shock protein 70. Molecular cell 36, 15-27]. However, recent evidence suggests that the PES interaction with Hsp70 is through non-specific interactions [Schlecht et al. (2013). Functional analysis of Hsp70 inhibitors. PloS one 8, e78443]. The molecule promotes caspase-dependent cell death only in tumor cells, suggesting some specificity to Hsp70i in vitro, although p53 binding has also been shown, which may explain its antitumor actions [Leu et al., 2009]. Moreover, MKT-077 and PES have potential reactive groups that render them covalent modifiers, which may contribute to side effects in vivo. YK5 is an allosteric inhibitor of Hsp70, recently identified using modeling techniques, but this molecule does not discriminate between Hsp70i and Hsc70 [Rodina et al., 2013].
Accordingly, there exists a need for receptor ligands selective for Hsp70i.